High-dose carboplatin-irinotecan-temozolomide is an effective salvage chemotherapy for relapsed or refractory neuroblastoma.

There is no clear consensus on the most effective treatment for relapsed/refractory high-risk neuroblastoma (NB). We retrospectively assessed seven NB patients with relapsed/refractory disease who received high-dose carboplatin-irinotecan-temozolomide (HD-CIT). Five of seven patients showed favorable therapeutic response (complete remission or partial remission). Regarding toxicity, the cytopenia period tended to prolong when more than three cycles were repeated, but nonhematological toxicities were controllable with general supportive care. Due to its antitumor efficacy and well-tolerated nonhematologic toxicity, HD-CIT is a promising salvage chemotherapy for relapsed/refractory NB. However, it is important to pay attention to the exacerbation of hematological toxicity when repeating the regimen.

Use of Cabozantinib to Treat MET -amplified Pediatric Colorectal Cancer.

Pediatric colorectal cancer (CRC) is extremely rare, with little information about genetic profiles compared with adult CRC. Here, a 13-year-old male with advanced CRC underwent cancer gene panel testing, which detected 4 genetic abnormalities ( MET amplification in addition to TP53 , SMAD4 , and CTNNA1 mutations) that might be associated with a poor prognosis. Based on high-level MET amplification, he received a multikinase inhibitor, cabozantinib, after failure of first-line and second-line chemotherapy, resulting in transient disease stabilization. Tailored targeted therapy based on molecular profiling can be an effective treatment strategy for rare cancers such as pediatric CRC.

RUNX1-Survivin Axis Is a Novel Therapeutic Target for Malignant Rhabdoid Tumors.

Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric malignancy with no effective therapy. Therefore, it is necessary to identify a target for the development of novel molecule-targeting therapeutic agents. In this study, we report the importance of the runt-related transcription factor 1 (RUNX1) and RUNX1-Baculoviral IAP (inhibitor of apoptosis) Repeat-Containing 5 (BIRC5/survivin) axis in the proliferation of MRT cells, as it can be used as an ideal target for anti-tumor strategies. The mechanism of this reaction can be explained by the interaction of RUNX1 with the RUNX1-binding DNA sequence located in the survivin promoter and its positive regulation. Specific knockdown of RUNX1 led to decreased expression of survivin, which subsequently suppressed the proliferation of MRT cells in vitro and in vivo. We also found that our novel RUNX inhibitor, Chb-M, which switches off RUNX1 using alkylating agent-conjugated pyrrole-imidazole polyamides designed to specifically bind to consensus RUNX-binding sequences (5'-TGTGGT-3'), inhibited survivin expression in vivo. Taken together, we identified a novel interaction between RUNX1 and survivin in MRT. Therefore the negative regulation of RUNX1 activity may be a novel strategy for MRT treatment.

Sensitive detection of GATA1 mutations using complementary DNA-based analysis for transient abnormal myelopoiesis associated with the Down syndrome.

INTRODUCTION: GATA1 mutation plays an important role in initiating transient abnormal myelopoiesis (TAM) and in the clonal evolution towards acute megakaryoblastic leukaemia (AMKL) associated with Down syndrome (DS). This study aimed to develop and validate the clinical utility of a complementary DNA (cDNA) analysis in parallel with the conventional genomic DNA (gDNA) Sanger sequencing (Ss), as an initial screening test for GATA1 mutations. METHODS: GATA1 mutations were evaluated using both gDNA and cDNA in 14 DS patients using Ss and fragment analysis (FA), respectively. RESULTS: The detection sensitivity of conventional gDNA sequencing was limited in low blast percentage TAM (LBP-TAM); however, cDNA-based Ss readily detected all the pathognomonic GATA1 mutations. The cDNA-based FA readily detected GATA1 frameshift mutation with a reliable sensitivity ranging from 0.005% to 0.01% of clonal cells. CONCLUSIONS: GATA1 mutations are heterogeneous; therefore, we would like to propose a dual cDNA and gDNA analysis as a standard diagnostic approach, especially for LBP-TAM. cDNA-based FA promises an excellent sensitivity for detecting frameshift GATA1 mutations in the longitudinal clonal evolution towards AMKL without using a patient specific primer.

Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy.

In vitro anti-inflammatory and anti-lipid accumulation properties of taxifolin-rich extract from the Japanese larch, Larix kaempferi.

The Japanese larch, (Larix kaempferi) is known to contain abundant taxifolin (dihydroquercetin) in its xylem. In this study, to assess the bioactivities of taxifolin rich methanol extract of L. kaempferi (LK-ME), anti-inflammatory effect, and the anti-lipid accumulation effect of LK-ME were investigated. The results showed that nitric oxide (NO) and reactive oxygen species (ROS) were reduced after treatment with LK-ME, and that lipid accumulation in adipocyte differentiated 3T3-L1 cells was inhibited after the cells were grown in medium containing LK-ME. Taxifolin, the major compound contained in LK-ME, and its related compounds, quercetin and luteolin also exhibited similar effects with LK-ME. The LK-ME exhibits relatively strong anti-inflammatory and anti-lipid accumulation activities compared with that of similar amounts of taxifolin contained in LK-ME, suggesting that other minor compounds contained in LK-ME is involved in the effects. These results indicate the potential of taxifolin-rich L. kaempferi extract for use as a supplement to prevent excess inflammation and obesity.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice.

Although the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin, a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients.

Influence of post-transplant mucosal-associated invariant T cell recovery on the development of acute graft-versus-host disease in allogeneic bone marrow transplantation.

Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are T cell subpopulations that possess innate-like properties. We examined the impact of post-hematopoietic stem cell transplantation (HSCT) MAIT and iNKT cell recovery on the clinical outcomes of 69 patients who underwent allogeneic HSCT at Kyoto University Hospital. Multivariate analyses identified the absolute number of MAIT cells (< 0.48/µL on day 60 post-HSCT) as the sole independent risk factor for grade I-IV and grade II-IV acute graft-versus-host disease (aGVHD) among patients who underwent bone marrow transplantation; no correlation was observed between post-HSCT iNKT cell recovery and the development of aGVHD. Six of the 15 patients in the MAIThigh (≥ 0.48/µL) group developed aGVHD, five within the first 30 days post HSCT. In contrast, 13 of the 15 patients in the MAITlow (< 0.48/µL) group developed aGVHD, seven after day 30 post HSCT. The overall survival of the MAITlow group was slightly shorter than that of the MAIThigh group. Thus, the post-HSCT recovery of MAIT cells is closely related to the development of delayed onset aGVHD and the outcome of post-HSCT, suggesting its utility for identifying a subset of patients that requires more prolonged and/or intense GVHD prophylaxis.

Childhood cancer survivors: Anxieties felt after treatment and the need for continued support.

BACKGROUND: Childhood cancer survivors (CCS) and their family members continue to live in fear even after treatment is concluded due to concerns about late effects and recurrences. The consequent long-term psychological burden requires long-term follow up suited to the anxieties and needs of CCS, hence the need for the present survey. METHODS: We conducted a questionnaire survey at medical facilities in the Chugoku and Shikoku regions of Japan with CCS who had survived for at least 5 years following treatment, and their family members. RESULTS: A total of 30 CCS (53%) and 27 CCS family members (47%) answered the questionnaires. The median age of the CCS and their family members (CCS parents) was 23 years and 51.5 years, respectively. The most common diagnosis was acute lymphoblastic leukemia (47%) and the median length of follow up after the conclusion of treatment was 11 years. The percentage of participants who responded that they knew about late effects was significantly lower among CCS than among CCS parents. Almost no significant difference was observed between CCS and CCS parents regarding anxieties at specific life stages. The main consultants for CCS and CCS parents were their family, but they sought opportunities for casual consultation for current worries outside the family. CONCLUSIONS: It is necessary for medical facilities not only to provide medical support, but also to establish a place where they can provide centralized consultation for the anxieties of CCS and their parents.

Impact of post-transplant minimal residual disease on the clinical outcome of pediatric acute leukemia.

This retrospective study examined the clinical significance of FCM-MRD in 36 patients with ALL and 29 patients with AML after their first allogeneic HSCT. Hematological (FCM-MRD ≥5.0%) and molecular relapse (FCM-MRD <5.0%) were first detected in 10 and two patients with ALL and in seven and eight patients with AML, respectively. Eight of 10 patients with molecular relapse eventually progressed to hematological relapse, although most were treated with immunological intervention by aggressive discontinuation of immunosuppressive therapy or donor lymphocyte infusion. Among these 12 patients, four of seven patients that obtained MRDneg CR following post-transplant chemotherapy remain alive and disease-free after their second HSCT; however, all five patients who underwent a second HSCT in non-CR died of disease or treatment-related complications. As the FCM-MRD monitoring system used in the current study was probably not sensitive enough to detect MRD, which could be elucidated by immunological intervention, more sensitive diagnostic tools are mandatory for post-transplant MRD monitoring. Additional studies are required to address the impact of presecond transplant MRD on the clinical outcome of second HSCT.

Impact of pretransplant minimal residual disease on the post-transplant outcome of pediatric acute lymphoblastic leukemia.

There are few reports on the clinical significance of MRD before HSCT in pediatric ALL. We retrospectively analyzed the clinical significance of FCM-based detection of MRD (FCM-MRD) before allogeneic HSCT in pediatric ALL. Of 38 pediatric patients who underwent allogeneic HSCT for the first time between 1998 and 2014, 33 patients were in CR and five patients were in non-CR. The CR group was further divided into two groups based on the pretransplant FCM-MRD level: the MRD(neg) (<0.01%; 30 patients) group and the MRD(pos) (≥0.01%; three patients) group. There were significant differences in the three-yr event-free survival rates between the CR and non-CR group, and between the MRD(neg) and MRD(pos) group. The three-yr cumulative RI in the MRD(neg) group were 27.3% ± 8.8%, whereas two of the three patients in the MRD(pos) group relapsed within one yr after HSCT. The clinical outcome of the MRD(pos) group was as poor as that of the non-CR group in pediatric ALL. Therefore, an improvement in pretransplant treatment that aims to achieve a more profound remission would contribute to reducing the risk of relapse.

Stimulation of macrophages with the ß-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A ß-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a ß-(1,3)-linked main chain with ß-(1,6)-linked glucose side residues. Various ß-glucans consisting of ß-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial ß-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Stimulation with the Aureobasidium pullulans-produced ß-glucan effectively induces interferon stimulated genes in macrophage-like cell lines.

A ß-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-ß mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-α receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-α stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-α was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation.

Relevance of signaling molecules for apoptosis induction on influenza A virus replication.

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.

ß-Glucan derived from Aureobasidium pullulans is effective for the prevention of influenza in mice.

ß-(1→3)-D-glucans with ß-(1→6)-glycosidic linked branches produced by mushrooms, yeast and fungi are known to be an immune activation agent, and are used in anti-cancer drugs or health-promoting foods. In this report, we demonstrate that oral administration of Aureobasidium pullulans-cultured fluid (AP-CF) enriched with the ß-(1→3),(1→6)-D-glucan exhibits efficacy to protect mice infected with a lethal titer of the A/Puerto Rico/8/34 (PR8; H1N1) strain of influenza virus. The survival rate of the mice significantly increased by AP-CF administration after sublethal infection of PR8 virus. The virus titer in the mouse lung homogenates was significantly decreased by AP-CF administration. No significant difference in the mRNA expression of inflammatory cytokines, and in the population of lymphocytes was observed in the lungs of mice administered with AP-CF. Interestingly, expression level for the mRNA of virus sensors, RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated protein 5) strongly increased at 5 hours after the stimulation of A. pullulans-produced purified ß-(1→3),(1→6)-D-glucan (AP-BG) in murine macrophage-derived RAW264.7 cells. Furthermore, the replication of PR8 virus was significantly repressed by pre-treatment of AP-BG. These findings suggest the increased expression of virus sensors is effective for the prevention of influenza by the inhibition of viral replication with the administration of AP-CF.

A small scale study on the effects of oral administration of the ß-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves.

BACKGROUND: The ß-(1 → 3),(1 → 6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the ß-(1 → 3),(1 → 6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves. FINDINGS: Holstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the ß-(1 → 3),(1 → 6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF. CONCLUSIONS: Our data indicated the possibility that oral administration of the ß-(1 → 3),(1 → 6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of ß-(1 → 3),(1 → 6)-D-glucans on domestic animals.